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EXPLORING THE UTILIZATION OF CRITHIDIA FASCICULATA AS A MODEL ORGANISM TO STUDY PATHOGENIC KINETOPLASMIDS Wakisa Kipandula A Thesis Submitted for the Degree of MPhil at the University of St Andrews 2017 Full metadata for this item is available in St Andrews Research Repository at: http://research-repository.st-andrews.ac.uk/ Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/12217 This item is protected by original copyright Exploring the utilization of Crithidia fasciculata as a model organism to study pathogenic kinetoplastids Wakisa Kipandula This thesis is submitted in partial fulfilment for the degree of MPhil at the University of St Andrews Date of Submission May 2017 i Abstract This study aimed to explore the utilization of C. fasciculata as a convenient model organism to study the cell biology and drug discovery vehicle of the pathogenic kinetoplastids. We specifically aimed to: (i) develop and validate aprotein A-TEV-protein C (PTP) tagged protein expression system for C. fasciculata, (ii) develop a Resazurin-reduction viability assay with C. fasciculata and use this for subsequent screening for anti-crithidial compounds from the GSK open access pathogen boxes, and (iii) to study the effects of ionizing gamma radiation on C. fasciculata. We report the construction of plasmid pNUS-PTPcH, which can be utilised to express PTP tagged kinetoplastids proteins in C. fasciculata for subsequent purification. As a proof of concept, we have shown that C. fasciculata can be efficiently transfected with this plasmid and facilitate the isolation of two protein complexes: replication factor C (RFC) and the exosome. We have demonstrated that the expressed PTP tagged-replication factor C subunit 3 (PTP-RFC3) co-purifies with RFC1, RFC2, RFC4, RFC5 and RAD17, and that the PTP tagged exosome subunit RRP4 co-purifies with RRP6, EAP1, RRP45, RRP40, RRP41B, CSL4, EAP2, RRP41A and EAP4. In addition, this thesis reports the development of a resazurin-reduction cell viability assay in C. fasciculata and reveals attractive core chemical scaffolds present in more than one of the open access GSK pathogen boxes, which will be followed up against the actual pathogenic kinetoplastids. Furthermore, this study has demonstrated that compared to cultured forms of T. cruzi which undergo growth arrest for 96 hours after exposure to 500 Gy of gamma radiation, C. fasciculata is able to recover and resume normal growth within 24 hours after being subjected to doses as high as 1000Gy. The constructed plasmid, the identified chemical scaffolds and the observed responses of C. fasciculata to gamma irradiation will help facilitate further studies aimed to discover novel drugs for kinetoplastid diseases. ii Declaration I, Wakisa Kipandula, hereby certify that this thesis, which is approximately 30,000 words in length, has been written by me, and that it is the record of work carried out by me, with the exception of a single plasmid consruction carried out by Dr Stuart MacNeill, and that it has not been submitted in any previous application for a higher degree. I was admitted as an MPhil student in November 2014. The work was carried out in University of St Andrews and the University Of Malawi College Of Medicine between 2014 and 2016. Date: 10th November 2017 Signature of candidate: We hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriate for the degree of MPhil in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree. Date Signatures of supervisors: iii In submitting this thesis to the University of St Andrews, I understand that I am giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby. I also understand that the title and the abstract will be published, and that a copy of the work may be made and supplied to any bona fide library or research worker, that my thesis will be electronically accessible for personal or research use unless exempt by award of an embargo as requested below, and that the library has the right to migrate my thesis into new electronic forms as required to ensure continued access to the thesis. I have obtained any third-party copyright permissions that may be required in order to allow such access and migration, or have requested the appropriate embargo below. The following is an agreed request by candidate and supervisor regarding the electronic publication of this thesis: (i) Access to printed copy and electronic publication of thesis through the University of St Andrews. Date: 10th November 2017 Signature of candidate: Date Signatures of supervisors: iv Acknowledgements I thank my supervisors Dr Stuart MacNeill and Professor Terry Smith for their valuable guidance, support, advice and motivation while working on this highly interesting project. I acknowledge the assistance offered to me by staff and fellow students in the Biomedical Sciences Research Complex, level 3. Special thanks to Wilberforce Sabiiti, Beatrice Mwagomba, Kondwani Katundu, Daniel Khomba and everybody who made my stay in very cold and boring St Andrews worthwhile. You are such a wonderful people. I am indebted to E. Tetaud for providing C. fasciculata pNus plasmids. This work would not have been possible without generous funding through the Global Health Implementation Programme at the University of St Andrews by the Gloag Foundation and the Western Union Foundation. It is with immense gratitude that I acknowledge the management of the University Of Malawi College Of Medicine for granting me a study leave. I am grateful to my wife Wezzie for her encouragement and perseverance while I was away. You are such a strong woman. Last but not least, the one above all of us, the omnipresent God for answering my prayers and for giving me the strength to plod on despite my constitution wanting to give up this work. Thank you so much Dear Lord. This work is dedicated to my son Andile. v Publications Some of the results from this thesis have been published in a well-recognised scientific journal as follows. i. Kipandula W, Smith TK, MacNeill SA. Tandem affinity purification of exosome and replication factor C complexes from the non-human infectious kinetoplastid parasite Crithidia fasciculata. Molecular and Biochemical Parasitology 217: 19-22 (2017). Available from, DOI: 10.1016/j.molbiopara.2017.08.004 vi Table of contents Abstract ................................................................................................................................................. ii Declaration ........................................................................................................................................... iii Acknowledgements ............................................................................................................................... v Publications…………………………………………………………………………………………………………………………….……….vi List of Abbreviations ............................................................................................................................ x 1. Chapter 1: Background introduction ......................................................................................... 1 1.1 Introduction ............................................................................................................................ 1 1.2 Current treatment for disease caused by kinetoplastids .......................................................... 4 1.3 Challenges to research on pathogenic kinetoplastids ............................................................. 6 1.4 C. fasciculata as a model organism to study kinetoplastid biology ........................................ 7 1.5 Aims of the study ................................................................................................................... 9 2. Chapter 2: Developing and validating an expression vector for subsequent isolation of PTP tagged kinetoplastids proteins in C. fasciculata ............................................................................... 10 2.1 Introduction .......................................................................................................................... 10 2.1.1 Kinetoplastids proteins expression systems .................................................................. 10 2.1.2 Multi protein complexes ............................................................................................... 11 2.1.3 Overview of protein complexes purification methods .................................................. 17 2.2 Materials and methods .......................................................................................................... 21 2.2.1 Organisms and reagents ................................................................................................ 21 2.2.2 General Molecular biology techniques ......................................................................... 21 2.2.3 Construction of the expression vector (pNUS-PTPcH) ................................................ 23 2.2.4 Identification of C. fasciculata and T. brucei homologous proteins from yeast RFC and exosome complexes ...................................................................................................................... 23 2.2.5 Cloning of the target subunits ......................................................................................
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